1. Field of the Invention
A process for the fermentative preparation of L-ornithine and microorganisms characterized by an increased export of the amino acid.
2. Description of the Related Art
L-Ornithine is known for its stimulatory action regarding liver function and is frequently utilized as an ingredient of medicaments and in sports nutrition.
L-Ornithine is nowadays prepared by various processes. One method is the fermentative preparation with the aid of microorganisms. Another method is alkaline hydrolysis of arginine, for example with barium hydroxide (CN 1594282 A). Another method is the biotransformation of arginine by immobilized microorganisms possessing an arginase activity (KR589121B1). A method of preparing L-ornithine from L-citrulline has also been described in the patent literature (JP 42007767 B4).
Microorganisms which are distinguished in that they eliminate L-ornithine into the culture medium have been described in the literature. Examples of said microorganisms are bacteria of the genus Corynebacterium, Brevibacterium, Bacillus (JP 43010996 B4, JP 57041912 B), Escherichia (U.S. Pat. No. 3,668,072 A), Providencia (JP 03195494) or Arthrobacter (U.S. Pat. No. 3,574,061).
L-Ornithine-producing microorganisms are often distinguished by being auxotrophic for the amino acids L-arginine or L-citrulline (described for Brevibacterium, Bacillus, Corynebacterium in EP 392708 B1 and KR 161147 B1 and for Escherichia in U.S. Pat. No. 366072 A). Furthermore, microorganisms have been described which are resistant to 2-thiazole-alanine, sulphaguanidine or 2-fluoropyruvate (Japanese Open-Laid publication No. 61-119194). EP 0393708 B1 describes L-ornithine producers which are distinguished by a lower resistance to ornithole and mycophenolic acid. Said properties may also be in a combined form.
The release of basic amino acids such as L-lysine, L-arginine and L-ornithine by way of passive diffusion from the cell is very poor (Bellmann et al. (Microbiology 2001; 147: 1765-74)). This has been well described for lysine by way of example. Vrlijc et al. (Journal of Bacteriology 1995; 177(14): 4021-7) have studied a plurality of export-deficient Corynebacterium glutamicum mutants. For one mutant, an intracellular concentration of 174 mM L-lysine was measured, while a value of only 0.7 mM was measured extracellularly.
Vrlijc et al. (Molecular Microbiology 1996; 22(5): 815-26 and Journal of Molecular Microbiology and Biotechnology 1999; 1: 327-336) and EP 0868527 B1 identified and described a novel exporter as L-lysine exporter (LysE). A defined LysE null mutant was no longer capable of transporting L-lysine out of the cell. The polypeptide encoded by the lysE gene is 233 amino acids or amino acid residues in length and is represented in SEQ ID No. 2.
After overexpression of the lysE gene in a lysine producer, an increase in L-lysine elimination was found.
Von Bellmann et al. (Microbiology 2001; 147: 1765-74) have characterized in more detail the LysE exporter with regard to the transport of various basic amino acids in C. glutamicum. The authors demonstrated that the transporter specifically exports the amino acids L-lysine and L-arginine out of the cell. The authors furthermore investigated whether LysE also exports L-ornithine out of the cell. For this purpose, first of all an L-arginine-auxotrophic C. glutamicum strain referred to as ATCC13032::argF was prepared.
The strain was cultured in 50 ml (batch culture) of a minimal medium referred to as CGXII which contained 40 g/l glucose. After an incubation period of 24 hours 60 mM L-ornithine, corresponding to 7.9 g/l, were measured. Intracellularly, an L-ornithine concentration of approx. 200 mM was measured in the cells of said strain over an incubation period of approx. 70 minutes. In order to clarify, whether LysE also transports L-ornithine out of the cell, the strain 13032::argF was transformed with the replicative plasmid pEC7lysE. This measure was intended to provide the strain with an increased LysE activity, thereby allowing the strain to transport L-ornithine into the medium at a higher rate of export. However, said measure did not increase the rate of L-ornithine export. The same rate of export (0.6 nmol min−1 (mg of dry mass)−1) was determined both for the control strain (13032::argF) and in the transformant (13032::argF, harbouring pEC7lysE). From this the authors concluded that L-ornithine is not exported by the LysE exporter. They furthermore drew the conclusion that there must be another, unknown export function (export protein) for L-ornithine in Corynebacterium glutamicum (Bellmann et al., 2001, page 1771, FIG. 5b) and page 1772, lines 21-28).
A variant LysE (see SEQ ID No. 4) was identified in C. glutamicum R, which differs from the amino acid sequence of the LysE exporter from strain ATCC 13032, depicted in SEQ ID No. 2, by an N terminus extended by three amino acid residues. The sequence of said amino acid residues is: methionine, valine, isoleucine (MVI). This LysE polypeptide from strain R has been described in EP 1266966 B1 as a variant which differs from the wild-type protein in the formation of a loop region or, more specifically, can no longer form said loop, and is therefore able to accomplish improved export of L-lysine and L-arginine.
Another LysE variant has been described by Gunji and Yasueda (Journal of Biotechnology 127, 2006, 1-13). The authors were interested in L-lysine formation by the obligately methylotrophic bacterium, Methylophilus methylotrophus. They transformed M. methylotrophus with a plasmid referred to as pSE which contained the C. glutamicum ATCC13869 lysE gene in order to improve lysine formation by M. methylotrophus. However, the authors found that they were able to establish only a mutated form of the lysE gene (lysE24) in a stable manner in M. methylotrophus. The open reading frame of the lysE gene has been shifted in the lysE24 allele due to the insertion of a thymine residue, resulting in the termination of the reading frame after 432 bp. The truncated reading frame codes for a LysE protein which is shorter by 92 aa residues at the C terminus than the wild-type LysE protein of C. glutamicum ATCC13869. It is 141 amino acid residues in length. In addition, the last 6 C-terminal amino acids of the truncated protein (residues 135-141) differ from the amino acids of the wild-type LysE amino acid sequence. An M. methylotrophus strain carrying the modified LysE allele on a plasmid (pSE24) was tested for lysine formation. To this end, the strain was assayed in 0.3 l of a minimal medium referred to as SEIIc in the form of a fed-batch culture for 50 hours. The authors found that the transformant also formed small quantities (0.07 mM corresponding to 11.8 mg/l) of L-ornithine, in addition to 0.55 mM L-lysine and 0.19 mM L-arginine. As explained by the authors, this observed formation of L-ornithine is due to either an altered substrate specificity of the mutated transporter or possibly the altered intracellular L-arginine pool of the strain. EP 1266966 Bi (inventors: Gunji and Yasueda) describes the positive action of the LysE24 transporter on the elimination of L-lysine and L-arginine.